CELL SORTING AND ANTIBODIES TO NEUROENDOCRINE CELLS

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The long term objective of this research is to increase understanding of the role of the cell surface in development and function of neuroendocrine cells, specifically hypothalamic magnocellular neurosecretory neurons. The primary aim is to determine if there are plasma membrane molecules which distinguish populations of neurosecretory cells and to utilize any such molecules to produce cell-type-specific markers. This question will be addressed by using monoclonal antibodies (MAbs) as probes for antigenic determinants in the plasmalemma. Because such molecules no doubt represent a very small proportion of the total antigenic molecules in extracts of neural tissue, several novel techniques are proposed to enhance the probability of obtaining MAbs against them. Two general strategies will be employed; enrichment of immunogens in neurosecretory cell surface molecules, and selection for activated lymphocytes prior to fusion with myeloma partners. Both strategies take advantage of fluorescence activated cell sorting (FACS) technology. Successful production of such MAbs would make it possible to test several specific hypotheses. Among these are: (1) that specific cell surface molecules mediating strict chemoaffinity mechanisms play a more important role than competitive rearrangement of circuitry during hypothalamic-neurohypophysial development; (2) that glucocorticoids and other hormonal or trophic factors influence cell survival and/or peptide gene expression during neurosecretory differentiation; and (3) that alterations in the concentrations of plasmalemmal molecules mediate the marked plastic changes which occur in the mature magnocellular neurosecretory system. A technical aim of this research is to apply novel FACS techniques to analysis of neuroendocrine cells. Methods have been developed for purification of rare neuronal populations (fixed neurophysin-containing neurons) for use as immunogens in attempts to produce MAbs against their surface antigens. In addition, use of the FACS as a tool for pre-fusion selection of activated lymphocytes is being explored in order to increase the probability of producing MAbs to rare antigens. Results of this research should increase our understanding of the role of the cell surface in normal ontogeny and function of neurosecretory neurons. In addition, they might shed light on such pathological states as idiopathic diabetes insipidus.